Patent
The present invention relates to an immunogenic and a non-toxic glycoconjugate comprising Bordetella pertussis LOS-derived oligosaccharide and pertussis toxin, a method of preparing such glycoconjugate, the pharmaceutical composition, a vaccine composition containing such glycoconjugate, and an application of the glycoconjugate. The glycoconjugate is prepared as a vaccine component for protection against infections caused by Bordetella pertussis.
1. A method for preparing a glycoconjugate comprising a B. pertussis lipooligosaccharide (LOS)-derived oligosaccharide (OS) or an oligosaccharide fragment of the B. pertussis LOS, and the pertussis toxin (PT) wherein the method comprises:
a. culturing of B. pertussis;
b. isolation of a B. pertussis LOS-derived OS or an OS fragment of the B. pertussis LOS from the cultured B. pertussis;
c. isolation of PT from B. pertussis culture medium;
d. activation of the B. pertussis LOS-derived OS or the OS fragment of the B. pertussis LOS;
e. conjugation of the activated B. pertussis LOS-derived OS or the activated OS fragment of the B. pertussis LOS, and PT by covalent linking of the activated B. pertussis LOS-derived OS or the activated OS fragment of the B. pertussis LOS, to free amino groups of lysine residues of pertussis toxin at pH=9 to provide a glycoconjugate; and
f. purification of the glycoconjugate;
wherein the content of the B. pertussis LOS-derived OS or the OS fragment of the B. pertussis LOS in the glycoconjugate with PT is 30-50% of the total weight of the glyconjugate.
2. The method according to claim 1 characterized in that the B. pertussis is cultured in Stainer-Scholte liquid medium with addition of (2,6-di-O-methyl)-B-cyclodextrin.
3. The method according to claim 1 , characterized in that the B. pertussis LOS-derived OS or the OS fragment of the B. pertussis LOS is activated with sodium periodate.
4. The method according to claim 1 , characterized in that the conjugation reaction in e) is a reductive amination reaction.
5. The method according to claim 1 , characterized in that the conjugation in e) is carried out in 0.2 M borate buffer.
6. A glycoconjugate comprising the B. pertussis LOS-derived OS or the OS fragment of the B. pertussis LOS, and the PT, wherein the glycoconjugate is made by the method of claim 1 .
7. A pharmaceutical composition comprising the glycoconjugate according to claim 6 and a pharmaceutically acceptable carrier.
8. A vaccine composition comprising the glycoconjugate according to claim 6 and a pharmaceutically acceptable carrier and optionally an adjuvant.
9. A glycoconjugate comprising the B. pertussis LOS-derived OS or the OS fragment of the B. pertussis LOS, directly linked by a covalent bond to free amino groups of lysine residues of PT, wherein the content of the B. pertussis LOS-derived OS or the OS fragment of the B. pertussis LOS in the glycoconjugate with PT is 30-50% of the total weight of the glyconjugate.
10. The glycoconjugate according to claim 9 , characterized in that the B. pertussis LOS-derived OS or the OS fragment of the B. pertussis LOS is selected from the group consisting of: a core oligosaccharide (incomplete glycoform, R) of B. pertussis LOS, a distal oligosaccharide of B. pertussis LOS, and the OS fragment of the B. pertussis LOS.
11. A vaccine composition comprising the glycoconjugate according to claim 9 and a pharmaceutically acceptable carrier and optionally an adjuvant.
12. The glycoconjugate according to claim 9 , characterized in that the B. pertussis LOS-derived OS or the OS fragment of the B. pertussis LOS is selected from the group consisting of oligosaccharides of
13. A method of treating a Bordetella pertussis infection, comprising administering the pharmaceutical composition as defined in claim 7 to a subject in need thereof.
14. A method of inducing an immune response against B. pertussis , comprising administering the vaccine composition as defined in claim 8 to a subject.
15. A method of treating a Bordetella pertussis infection, comprising administering the vaccine composition as defined in claim 8 to a subject in need thereof.
16. A method of inducing an immune response against B. pertussis , comprising administering the vaccine composition as defined in claim 11 to a subject.
17. A method of treating a Bordetella pertussis infection, comprising administering the vaccine composition as defined in claim 11 to a subject in need thereof.